Descrição
Septins are highly conserved proteins that are present in fungi and animals. They are essential for many cellular functions i.e., cell proliferation, phagocytosis, ciliogenesis, cell polarization and morphogenesis and others that require restructuring cytokinesis. (1) In the specific case of M. oryzae, septins play a critical role in the formation of a toroidal actin filament network at the base of the appressorium, enabling the penetration of the leaf surface. Research is needed to investigate the precise roles and contributions of septins 7 and 8 in the cellular processes and pathogenicity of M. Oryzae. The aim of this study is to structurally describe both the individual and collective M. Oryzae septins. The septins are isolated after being expressed in E. coli Rosetta (DE3) and then analyzed for their stability, oligomerization, and nucleotide content. Crystallization and X-ray diffraction data collection are performed to resolve the structures, while negative contrast electron microscopy confirms complex organization. (2) Cryo-EM is employed for structural resolution of the complexes. We are currently able to express sep8-G for complex analysis while Sep-7 and Sep8-sumo and MBP is still unable to express. After purification of the sample by affinity chromatography (on a nickel column, the samples were purified by molecular exclusion chromatography (superdex200) and the chromatogram showed that Sep8-G in a volume that would correspond to the fact that they are forming Dimers. The denaturing SDS-PAGE gel showed that the septins are not being expressed equimolarly and modifications in conditions are also being made to improve stability and expression. Using the Gibson Assembly method (3) sub-clonings will be made to express the G domains of the septins: sep7G, sep8G and sep8GCDTM, these sub-clonings will be sequenced to confirm that the sequences are correct and to continue with the expression tests.
Referências
1 VALADARES, N. F.; PEREIRA, H. M.; ARAÚJO, A. P. U.; GARRATT, R. C. Septin structure and filament assembly. Biophysical Reviews, v. 9, n. 5, p. 481-500, Oct. 2017. DOI: http://dx.doi.org/10.1007/s12551-017-0320-4.
2 OSÉS-RUIZ, M. et al. Appressorium-mediated plant infection by Magnaporthe oryzae is regulated by a Pmk1-dependent hierarchical transcriptional network. Nature Microbiology, v. 6, n. 11, p. 1383-1397, 2021. DOI: http://dx.doi.org/10.1038/s41564-021-00978-w.
3 GIBSON, D. G. Enzymatic assembly of overlapping DNA fragments. In: VOIGT, C. (ed.) Synthetic biology part B: computer aided design and DNA assembly. San Diego: Academic Press, 2011. cap. 15, p. 349-361. (Methods in Enzymology, v. 498). DOI: https://doi.org/10.1016/B978-0-12-385120-8.00015-2.
| Certifico que os nomes citados como autor e coautor estão cientes de suas nomeações. | Sim |
|---|---|
| Palavras-chave | Magnaporthe oryzae. Crystallography. Electron Microscopy (EM). |
| Orientador e coorientador | Advisor Richard Charles Garratt |
| Subárea 1 | Biofísica |
| Subárea 2 (opcional) | Cristalografia |
| Agência de Fomento | CAPES |
| Número de Processo | 88887.801323/2023-00 |
| Modalidade | DOUTORADO |
| Concessão de Direitos Autorais | Sim |
